1. Field of the Invention
The field of the invention relates generally to the detection of nucleotide polymorphisms, and in particular to the discrimination of perfectly matched probe-target hybrids.
2. Related Art
Single nucleotide polymorphism (“SNP”) contributes to phenotypic diversity in human populations. Averaging about 1 in every 1000 nucleotide bases, SNP represents the most common type of sequence variation among individual genomes and have been found to play a significant role in a wide range of human diseases. Efficient schemes to detect SNPs are critical for applications ranging from disease gene mapping to clinical diagnostics.
Screening and identification of SNPs are central to many pharmaceutical companies' short term and long term research goals. The rationale is that people with particular SNPs are more likely than others to develop certain diseases. Knowledge about these SNPs can therefore lead to the development of novel therapeutic approaches for diseases such as hypertension, diabetes, and cancer. Thus, any technology that can great simplify and speed up the process of SNP discovery and characterization is valuable for therapeutic discovery efforts.
The intense interest in SNP data has led to multiple efforts to develop SNP screening and identification methods suitable for population studies. Currently, the gold standard is direct sequencing using one of the standard sequencing methods such as the dye-terminator cycle sequencing approach. However, despite significant advances in DNA sequencing technology, direct sequencing has not established itself as the method of choice for large scale SNP typing. In part, this is due to different scopes of focus—direct sequencing can provide up to 1 kilobase of nucleotide sequence at a time, while SNP screening is usually concerned with sequence information at a single base.
Recently, a variety of alternative high-throughput approaches focusing on the identification of nucleotides at specific positions of interest have been developed. In these approaches, detection is accomplished by monitoring the light emitted by SNP reaction products, measuring the mass of SNP reaction products, or detecting the cantilever beam deflection cause by SNP reaction products. However, these methods currently suffer from several drawbacks including the requirement for specialized personnel and the need for non-portable and relatively expensive equipment.
Fluorescently-labeled oligonucleotide probes known as molecular beacons have been demonstrated to detect single nucleotide variations in DNA sequences. These probes contain hairpin-stem structures that are necessary for detecting single base pair mismatches. Various groups have shown that molecular beacons can be used for SNP typing. However, current molecular beacon technology relies on laser and other optical equipment which are relatively expensive and bulky.
A well established technique for detecting nucleic acid sequences is sandwich DNA hybridization, in which a DNA target is sandwiched between a capture probe and a detector probe. In most implementations, the technique has been used with an enzyme-linked immunosorbent assay type system coupled to optical detection of output signal. The incorporation of enzyme offers the opportunity to amplify output signals. One way that output signals can be amplified is by catalyzed reporter deposition, as described in U.S. Pat. No. 5,196,306 of Bobrow, et al., hereby incorporated by reference. Catalyzed reporter deposition is a signal amplification scheme typically based on the deposition of hapten-labeled tyramide molecules through the creation of highly reactive, peroxidase-generated products. The deposited haptens can serve as binding targets for additional antibody-enzyme conjugates. To date, catalyzed reporter deposition has been reported only in conjunction with optical detection methods.
An alternative to hybrid detection by enzyme-linked immunosorbent methods is electrochemical detection, whereby an electrical sensor detects electrical currents generated by an enzyme acting on an electrochemically active substrate. Electrochemical detection permits signal amplification but without the bulkier and more costly devices potentially required of optical detection. However, no reported electrochemical method has been shown to contain sufficient specificity for detection of single base pair mismatches.
Throughput, accuracy, speed, and cost-effectiveness are among the most important criteria for evaluating an SNP typing method used for large-scale screening. What is needed is an SNP typing method that is quick, inexpensive and easy to perform.